HHS Public Access Author manuscript Author Manuscript
J Cutan Pathol. Author manuscript; available in PMC 2016 January 16. Published in final edited form as: J Cutan Pathol. 2015 November ; 42(11): 807–814. doi:10.1111/cup.12564.
Diagnostic utility of 5-hydroxymethylcytosine immunohistochemistry in melanocytic proliferations Nemanja Rodić1, John Zampella2, Reema Sharma1, Kathleen H. Burns1,3,4, and Janis M. Taube1,2,3 1Department
of Pathology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
Author Manuscript
2Department
of Dermatology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
3Department
of Oncology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
4The
High Throughput Biology Center, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
Abstract
Author Manuscript Author Manuscript
Decreased hydroxymethylated cytosine (5-hydroxymethycytosine, 5-hmC) is reported to correlate with melanocyte dysplasia. The purpose of this study was to assess the diagnostic utility of this observation. 5-hmC immunohistochemistry was performed on tissue microarrays containing 171melanocytic lesions from two different institutions. An immunohistochemical staining score representing the percentage and intensity of nuclear staining was assigned. The performance characteristics of 5-hmC immunohistochemistry for discriminating between a nevus and melanoma were determined. Additional cases of melanoma arising in a nevus (n = 8), nodal nevi (n = 5) and melanoma micrometastases to a lymph node (n = 6) were also assessed. Pronounced 5hmC loss was observed in melanomas when compared with nevi (mean ± standard deviation = 6.71 ± 11.78 and 55.19 ± 23.66, respectively, p < 0.0001). While the mean immunohistochemical staining score values for melanocytic nevi and melanoma were distinct, there was considerable variability in immunohistochemical staining score within a single diagnostic category. The sensitivity and specificity of this assay for nevus vs. melanoma is 92.74 and 97.78%, respectively. Distinct biphasic staining patterns were observed in cases of melanoma arising in association with a nevus. Relative changes of 5-hmC expression within a single lesion may be more informative than absolute values when using 5-hmC as a diagnostic adjunct.
Dr Janis M. Taube, MD, Department of Dermatopathology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Blalock 907, 600N. Wolfe St. Baltimore, MD 21287, USA, Tel: +410 955-3484, Fax: +410 955-2445, [email protected]. Author contribution All authors confirmed they have contributed meaningfully to the intellectual content of this publication, and this study was supported in part by the Resident Research Fund grant from the Department of Pathology at the Johns Hopkins Hospital (NR) and by the Fred and Janet Sanfilippo Research Fund (NR).
Rodić et al.
Page 2
Author Manuscript
Keywords 5-hydroxymethylcytosine; epigenetics; melanoma; nevus; dermatopathology
Author Manuscript
The classic model of melanomagenesis is that mutations initiate and promote tumor formation and progression. Indeed, the melanoma genome contains one of the highest somatically acquired nucleotide mutation frequencies of any human cancer.1 These mutations are dominated by C to T transitions, which are generated by ultraviolet A and B electromagnetic radiation.1 The mutations reproducibly occur in genes belonging to one of the three, near mutually exclusive, major oncogenic pathways that sustains proliferative signaling. In decreasing order of frequency, the most common mutations involve BRAF, NRAS and C-Kit genes.2–5 In addition to point mutations, chromosomal gains and losses are also prevalent in melanocytic tumors.6,7 It is now known that in addition to changes in the sequence of genomic DNA, epigenetic aberrations also play a role in melanoma formation.
Author Manuscript
Gene expression is tightly and dynamically regulated by a multi-layer epigenetic system that includes DNA methylation, histone modification and non-coding RNAs. Pathologic alterations in the epigenome are thought to contribute to tumor development and virulence (reviewed in8). For example, hypermethylation of the promoters of tumor suppressor genes may lead to gene silencing and condensation or opening of the chromatin structure via histone modification may alternately prevent or facilitate gene expression, depending on the type and position of the modification. In cutaneous melanoma, genes impacting melanoma progression and metastases such as those involving DNA repair, cell cycle regulation and invasion, have all been shown to be silenced by DNA hypermethylation.9–11 Demethylation, on the other hand, has been associated with the aberrant re-expression of certain genes, which are typically silenced early in development.12 His-tone modifications in melanoma are not as well understood, though there is evidence to suggest that the histone variant macroH2A is lost in melanoma relative to nevi.13 MacroH2A is associated with chromatin condensation and its loss results in an increased proliferative and migratory phenotype.
Author Manuscript
DNA methylation associated with gene regulation occurs at the 5-carbon position of the cytosine base, resulting in 5-methylcytosine (5-mC). It is a single step catalyzed by DNA methyltransferases. The demethylation process is more complicated and requires multiple steps, the first of which is performed by the Ten Eleven Translocase family enzymes and results in the formation of 5-hydroxymethycytosine (5-hmC).14,15 5-hmC is the most abundant intermediate product of the demethylation pathway, and recently Lian et al. described 5-hmC loss as an ‘epigenetic hallmark’ of melanoma.16 In their initial description, they showed that loss of 5-hmC was associated with higher stage lesions and worse prognosis in patients with melanoma.16 In a follow-up report, the authors went on to further define 5-hmc loss in specific-histopathologic subtypes of melanocytic lesions, whereby they demonstrated progressive loss of 5-mhC with increasing nuclear diameter in a spectrum of melanocytic lesions that included benign nevi, low-grade dysplastic nevi, high-grade dysplastic nevi and invasive melanoma.17 While the mean/median differences between 5hmC expression in different diagnostic categories were presented in these earlier reports, the diagnostic sensitivity and sensitivity of the 5-hmC immunohistochemical assay were not
J Cutan Pathol. Author manuscript; available in PMC 2016 January 16.
Rodić et al.
Page 3
Author Manuscript
determined. The purpose of this study was to characterize further the immunohistochemical assay for 5-hmC expression to determine its potential as a diagnostic adjunct in melanocytic lesions.
Materials and methods Specimen procurement and tissue microarray format
Author Manuscript
One hundred seventy one benign and malignant melanocytic lesions were gathered from two separate institutions for analysis. All tissue specimens were originally procured per routine surgical pathology protocol and arranged for analysis using tissue microarray technology. This study was approved by the review board at both institutions. The tissue microarray from our institution (cohort I) consisted of 43 melanocytic lesions that were acquired from 1974 to 2008, Table 1. Each specimen was tiled in technical triplicate or quadruplicate cores. Selected melanoma cases were tiled as biological replicates (i.e. primary vs. metastatic melanoma specimen from the same patient). The tissue microarray from the outside institution (cohort II) contained 128 melanocytic lesions. Each case was cored once and did not have associated clinical information. In addition to the tissue microarrays, whole mount sections of melanoma arising in association with a nevus (n = 8), micrometastatic melanoma to lymph nodes (n = 6) and nodal melanocytic nevi (n = 5) were stained for 5hmC expression. Pathologic assessment
Author Manuscript Author Manuscript
The immunohistochemical protocol used in this report was performed according to the previously optimized and validated method by Haffner et al.18 For each staining run, a positive control slide of representative tissue sections from primary prostatic carcinoma was included. The nuclei of the basal cell layer within benign prostatic glands are immunoreactive with 5-hmC antibody, whereas the nuclei of malignant glands are nonimmunoreactive with 5-hmC (data not shown). In each experimental tissue microarray core, at least 40 individual nuclei were counted to ensure uniform results. Forty nuclei per core cutoff were chosen because we determined that the relative coefficient of variation for 5hmC immunolabeling within any given core is
J Cutan Pathol. Author manuscript; available in PMC 2016 January 16. Published in final edited form as: J Cutan Pathol. 2015 November ; 42(11): 807–814. doi:10.1111/cup.12564.
Diagnostic utility of 5-hydroxymethylcytosine immunohistochemistry in melanocytic proliferations Nemanja Rodić1, John Zampella2, Reema Sharma1, Kathleen H. Burns1,3,4, and Janis M. Taube1,2,3 1Department
of Pathology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
Author Manuscript
2Department
of Dermatology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
3Department
of Oncology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
4The
High Throughput Biology Center, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD, USA
Abstract
Author Manuscript Author Manuscript
Decreased hydroxymethylated cytosine (5-hydroxymethycytosine, 5-hmC) is reported to correlate with melanocyte dysplasia. The purpose of this study was to assess the diagnostic utility of this observation. 5-hmC immunohistochemistry was performed on tissue microarrays containing 171melanocytic lesions from two different institutions. An immunohistochemical staining score representing the percentage and intensity of nuclear staining was assigned. The performance characteristics of 5-hmC immunohistochemistry for discriminating between a nevus and melanoma were determined. Additional cases of melanoma arising in a nevus (n = 8), nodal nevi (n = 5) and melanoma micrometastases to a lymph node (n = 6) were also assessed. Pronounced 5hmC loss was observed in melanomas when compared with nevi (mean ± standard deviation = 6.71 ± 11.78 and 55.19 ± 23.66, respectively, p < 0.0001). While the mean immunohistochemical staining score values for melanocytic nevi and melanoma were distinct, there was considerable variability in immunohistochemical staining score within a single diagnostic category. The sensitivity and specificity of this assay for nevus vs. melanoma is 92.74 and 97.78%, respectively. Distinct biphasic staining patterns were observed in cases of melanoma arising in association with a nevus. Relative changes of 5-hmC expression within a single lesion may be more informative than absolute values when using 5-hmC as a diagnostic adjunct.
Dr Janis M. Taube, MD, Department of Dermatopathology, Johns Hopkins University School of Medicine and Sidney Kimmel Comprehensive Cancer Center, Blalock 907, 600N. Wolfe St. Baltimore, MD 21287, USA, Tel: +410 955-3484, Fax: +410 955-2445, [email protected]. Author contribution All authors confirmed they have contributed meaningfully to the intellectual content of this publication, and this study was supported in part by the Resident Research Fund grant from the Department of Pathology at the Johns Hopkins Hospital (NR) and by the Fred and Janet Sanfilippo Research Fund (NR).
Rodić et al.
Page 2
Author Manuscript
Keywords 5-hydroxymethylcytosine; epigenetics; melanoma; nevus; dermatopathology
Author Manuscript
The classic model of melanomagenesis is that mutations initiate and promote tumor formation and progression. Indeed, the melanoma genome contains one of the highest somatically acquired nucleotide mutation frequencies of any human cancer.1 These mutations are dominated by C to T transitions, which are generated by ultraviolet A and B electromagnetic radiation.1 The mutations reproducibly occur in genes belonging to one of the three, near mutually exclusive, major oncogenic pathways that sustains proliferative signaling. In decreasing order of frequency, the most common mutations involve BRAF, NRAS and C-Kit genes.2–5 In addition to point mutations, chromosomal gains and losses are also prevalent in melanocytic tumors.6,7 It is now known that in addition to changes in the sequence of genomic DNA, epigenetic aberrations also play a role in melanoma formation.
Author Manuscript
Gene expression is tightly and dynamically regulated by a multi-layer epigenetic system that includes DNA methylation, histone modification and non-coding RNAs. Pathologic alterations in the epigenome are thought to contribute to tumor development and virulence (reviewed in8). For example, hypermethylation of the promoters of tumor suppressor genes may lead to gene silencing and condensation or opening of the chromatin structure via histone modification may alternately prevent or facilitate gene expression, depending on the type and position of the modification. In cutaneous melanoma, genes impacting melanoma progression and metastases such as those involving DNA repair, cell cycle regulation and invasion, have all been shown to be silenced by DNA hypermethylation.9–11 Demethylation, on the other hand, has been associated with the aberrant re-expression of certain genes, which are typically silenced early in development.12 His-tone modifications in melanoma are not as well understood, though there is evidence to suggest that the histone variant macroH2A is lost in melanoma relative to nevi.13 MacroH2A is associated with chromatin condensation and its loss results in an increased proliferative and migratory phenotype.
Author Manuscript
DNA methylation associated with gene regulation occurs at the 5-carbon position of the cytosine base, resulting in 5-methylcytosine (5-mC). It is a single step catalyzed by DNA methyltransferases. The demethylation process is more complicated and requires multiple steps, the first of which is performed by the Ten Eleven Translocase family enzymes and results in the formation of 5-hydroxymethycytosine (5-hmC).14,15 5-hmC is the most abundant intermediate product of the demethylation pathway, and recently Lian et al. described 5-hmC loss as an ‘epigenetic hallmark’ of melanoma.16 In their initial description, they showed that loss of 5-hmC was associated with higher stage lesions and worse prognosis in patients with melanoma.16 In a follow-up report, the authors went on to further define 5-hmc loss in specific-histopathologic subtypes of melanocytic lesions, whereby they demonstrated progressive loss of 5-mhC with increasing nuclear diameter in a spectrum of melanocytic lesions that included benign nevi, low-grade dysplastic nevi, high-grade dysplastic nevi and invasive melanoma.17 While the mean/median differences between 5hmC expression in different diagnostic categories were presented in these earlier reports, the diagnostic sensitivity and sensitivity of the 5-hmC immunohistochemical assay were not
J Cutan Pathol. Author manuscript; available in PMC 2016 January 16.
Rodić et al.
Page 3
Author Manuscript
determined. The purpose of this study was to characterize further the immunohistochemical assay for 5-hmC expression to determine its potential as a diagnostic adjunct in melanocytic lesions.
Materials and methods Specimen procurement and tissue microarray format
Author Manuscript
One hundred seventy one benign and malignant melanocytic lesions were gathered from two separate institutions for analysis. All tissue specimens were originally procured per routine surgical pathology protocol and arranged for analysis using tissue microarray technology. This study was approved by the review board at both institutions. The tissue microarray from our institution (cohort I) consisted of 43 melanocytic lesions that were acquired from 1974 to 2008, Table 1. Each specimen was tiled in technical triplicate or quadruplicate cores. Selected melanoma cases were tiled as biological replicates (i.e. primary vs. metastatic melanoma specimen from the same patient). The tissue microarray from the outside institution (cohort II) contained 128 melanocytic lesions. Each case was cored once and did not have associated clinical information. In addition to the tissue microarrays, whole mount sections of melanoma arising in association with a nevus (n = 8), micrometastatic melanoma to lymph nodes (n = 6) and nodal melanocytic nevi (n = 5) were stained for 5hmC expression. Pathologic assessment
Author Manuscript Author Manuscript
The immunohistochemical protocol used in this report was performed according to the previously optimized and validated method by Haffner et al.18 For each staining run, a positive control slide of representative tissue sections from primary prostatic carcinoma was included. The nuclei of the basal cell layer within benign prostatic glands are immunoreactive with 5-hmC antibody, whereas the nuclei of malignant glands are nonimmunoreactive with 5-hmC (data not shown). In each experimental tissue microarray core, at least 40 individual nuclei were counted to ensure uniform results. Forty nuclei per core cutoff were chosen because we determined that the relative coefficient of variation for 5hmC immunolabeling within any given core is
